人血管抑素K1-3在毕赤酵母胞内表达的研究

目的:构建人血管抑素K1-3的重组酵母胞内表达质粒PHIL-D2/K1-3,获得重组目的蛋白.方法:从胎儿肝脏组织用RT-PCR方法扩增人血管抑素K1-3目的基因,克隆至酵母胞内表达质粒PHIL-D2中,线性化后转化到毕赤酵母GS115胞内,经PCR和Sourthern杂交筛选出阳性转化子,并用甲醇诱导表达.表达产物进行SDS-PAGE(12%)和Western blot检测,经赖氨酸亲和层析柱纯化后,用Lowry法测定蛋白含量,并进行生物活性测定.结果:SDS-PAGE和Western blot结果显示,表达蛋白质的相对分子量为30kd左右,低糖基化.Lowry法测定蛋白表达量为6.8mg/L.活性测定实验表明重组血管抑素能特异抑制人血管内皮细胞的增殖.结论:在毕赤酵母GS115胞内成功表达了人血管抑素K1-3重组蛋白.

A Study on Intracellular Expresstion of Human Angiostatin K1 - 3 in Pichia Pastoris

Objective:To construct a recombinant plasmid PHIL-D2/K1-3 for the expression of human AngiostatinK1-3 in Pichia pastoris.Methods :The gene of human AngiostatinK1-3 was amplified from the embryo liver tissue using RT-PCR and then cloned into PHIL-D2 vector.After being linearized,the recombinant plasmid was transformed into Pichia pastoris GS115.The positive transformant was identified by PCR and Southern blotting and then induced by methanol.The expressed products was analyzed by SDS-PAGE(12%) and Western blot.After being purified by lysine/Sepharose-4B affinity chromatography,the recombinant protein concentration was determined with Lowry's method and its antiangiogenic activity was assayed as well.Results: The recombinant protein was about 30 KD.and the level of expression was 6.8mg/L.In the activity assay,the recombinant protein could strongly inhibit the proliferation of HUVEC.Conclution: The recombinant human AngiostatinK1-3 is successfully expressed in Pichia pastoris GS115.